Device for the amplification of dna, comprising a microwave energy source

ABSTRACT

The invention relates to a device for the amplification of DNA in a reaction mixture, the device ( 1 ) comprising a heated chamber ( 2 ) including a rotor ( 3 ) for holding a plurality of reaction vessels for reaction mixtures, a drive means for the rotor, a microwave energy source ( 8 ) with means for controlled delivery of said energy to the reaction mixtures, and a system ( 14 - 16 ) for determining denaturation of double-stranded DNA. The invention also provides a method for the amplification of a nucleic acid strand. In the first step of the method, a reaction mixture is formed comprising the target nucleic acid strand, nucleotides, a primer, a thermostable nucleic acid polymerase, and, if necessary, a reagent for the detection of denaturation of double-stranded DNA. In the second step, the mixture is incubated at a temperature which allows synthesis of a nucleic acid strand complementary to the target nucleic acid strand. The third step comprises denaturing double-stranded DNA formed in the second step by microwave energisation of the reaction mixture with monitoring of the mixture to determine the denaturation end point. The reaction mixture is allowed to cool to a temperature at which primer anneals to the target nucleic acid strand in the fourth step. The second to fourth steps are repeated until a desired level of amplification is achieved.

TECHNICAL FIELD

The invention the subject of this application relates to procedures thatrequire the rapid cycling of a reaction mixture between differentconditions. More particularly, the invention relates to a device andmethod for use in such cycling procedures. The device and method areparticularly suited for amplifying DNA in processes involving successivecycles of annealing, polymerisation and denaturation steps.

BACKGROUND ART

In a number of applications such as gene analysis and DNA profiling, itis desirable to multiply the amount of particular nucleic acid sequencespresent in a sample. For example, a duplex DNA segment of up toapproximately six thousand base pairs in length may be amplified manymillion fold by means of the polymerase chain reaction (PCR), startingfrom as little as a single copy. In this technique, a denatured duplexDNA sample is incubated with a molar excess of two oligonucleotideprimers, one being complementary to a short strand of the DNA duplex andthe other being identical to a second short sequence upstream of it(i.e., more 5′).

Each primer anneals to its complementary sequence and primes thetemplate-dependent synthesis by DNA polymerase of a complementary strandwhich extends beyond the site of annealing of the other primer throughthe incorporation of deoxynucleotide triphosphates. Each cycle ofdenaturation, annealing and synthesis affords an approximate doubling ofthe amount of target sequence, where the target sequence is defined asthe DNA sequence subtended by and including the primers. A cycle iscontrolled by varying the temperature to permit successive denaturationof complementary strands of duplex DNA, annealing of the primers totheir complementary sequences, and primed synthesis of new complementarysequences. The use of a thermostable DNA polymerase obviates thenecessity of adding new enzyme for each cycle, thus allowing automationof the DNA amplification process by thermal cycling. Twentyamplification cycles increases the amount of target sequence byapproximately one million-fold.

More detailed information regarding the polymerase chain reaction can befound in standard texts such as PCR Protocols—A Guide to Methods andApplication (M. A. Innis, D. H. Gelfard, J. J. Sainskey and T. J. Whiteed's, Academic Press, Inc., San Diego, 1990), the entire content ofwhich is incorporated herein by cross reference.

It has been found that the technique of DNA polymerization requiresrapid controlled heating and cooling cycles. The art is replete withincubators and other devices to achieve this end—see, for example,International Patent Application No. PCT/AU90/00560 (Publication No. WO91/07504) and International Patent Application No. PCT/AU98/00277(Publication No. WO 98/49340).

Typically, a device used for PCR consists of a heat conductive materialprovided with channels or cavities adapted to receive vessels in whichthe reaction is to take place, for example Eppendorf™ tubes. The heatconductive material is then provided with heating/cooling means.

Wittwer et al. in Biotechniques 10, 76-82 (1991) state that incommercial units for automated DNA amplification, temperature transitionrates are usually no less than 3° C. per second when metal blocks orwater are used for thermal equilibration and samples are contained inplastic micro-centrifuge tubes. A significant fraction of cycle time isspent heating and cooling the sample, as opposed to being spent atoptimal denaturation, annealing and elongation temperatures. Extendedamplification times of two to four hours are common and long transitiontimes make it difficult to determine optimal temperatures and times foreach stage. Instantaneous temperature changes are not possible becauseof sample, container and cycler heat capacities.

In the international application referred to above—PCT/AU98/00277, theentire content of which is incorporated herein by cross reference—thereis disclosure of a method and device for thermal cycling of reactionmixtures which at least partially overcome the temperature transitionrate problem referred to in the previous paragraph. In thePCT/AU98/00277 device and method, reaction mixture vessels are held in arotor which rotates in a controlled temperature environment. Temperaturecycling is effected by heating and cooling of the environment. Thisallows more efficient heating and cooling of the reaction mixtures sincetransition losses are minimised. Also, the fact that the samples arespinning ensures that all samples are heated and cooled at the samerate. Consequently, no equilibration times are required when a set pointtemperature is reached.

While the PCT/AU98/00277 device and method reduces cycle time, the timeis nevertheless too long for use in a linear amplification process. Sucha process is desirable as it allows amplification of a single DNA orcDNA strand. Other available amplification devices and methods similarlyhave cycle times of such a period that use for linear amplification isnot feasible.

It would therefore be desirable to have available a device and methodsuitable for linear amplification of a nucleic acid strand.

SUMMARY OF THE INVENTION

An object of the invention is to provide a device and method for thecycling of a reaction mixture in which the cycle time is such that itallows the device and method to be used for the linear amplification ofDNA.

According to a first embodiment of the invention, there is provided adevice for the amplification of DNA in a reaction mixture, the devicecomprising:

-   -   a heated chamber including a rotor for holding a plurality of        reaction vessels for reaction mixtures;    -   a drive means for said rotor;    -   a microwave energy source with means for controlled delivery of        said energy to said reaction mixtures; and    -   a system for determining denaturation of double-stranded DNA.

According to a second embodiment of the invention, there is provided amethod for the amplification of a nucleic acid strand, the methodcomprising the steps of:

-   i) forming a reaction mixture comprising said target nucleic acid    strand, nucleotides, a primer, a thermostable nucleic acid    polymerase, and, if necessary, a reagent for the detection of    denaturation of double-stranded DNA;-   ii) incubating said mixture at a temperature which allows synthesis    of a nucleic acid strand complementary to said target nucleic acid    strand;-   iii) denaturing double-stranded DNA formed in step (ii) by microwave    energisation of said reaction mixture with monitoring of said    mixture to determine the denatur-ation end point;-   iv) allowing said reaction mixture to cool to a temperature at which    primer anneals to said target nucleic acid strand; and-   v) repeating steps (ii) to (iv) until a desired level of    amplification is achieved.

It will be appreciated from the foregoing definitions of embodiments ofthe invention that the invention relies on microwave energy to denatureDNA rather than heat as in conventional procedures. In this way, fastercycles are possible as there is no need to externally heat reactionmixtures to denature DNA or to delay the primer annealing andpolymerisation steps while the mixture cools to the set temperature forprimer annealing and polymerisation.

With regard to the first embodiment of the invention, the device isessentially the same as that described in PCT/AU98/00277, but modifiedto include the microwave energy denaturisation feature. In broad terms,the device chamber can be any suitable, typically insulated, containerfor the internal device components and for association of ancillarycomponents therewith. The chamber advantageously has a lid or sealableopening for loading the device rotor.

Heating of the chamber can be by any of the means used for controlledheating of automated reaction devices. Typically, heating is by a heaterlocated within the chamber with circulation of heated air within thechamber aided by a fan. Alternatively, heated air can be supplied to thechamber from a port or ports in a chamber wall.

A device according to the invention will normally have a temperaturesensor within the chamber which is linked to an associated computerresponsible for controlling the operation of the device. Through sensingof the chamber temperature, heater operation can be controlled via thecomputer.

With regard to the device rotor, this can be any suitable rotor providedthat it is not fabricated from a metals material. Rotors are alsopreferably not heat conductive or electrically conductive. This is toavoid heating of the rotor during microwave energisation of reactionmixtures. Heat accumulation by the rotor would prevent maintenance ofthe reaction mixture at the annealing and polymerisation temperature.Advantageously therefore, rotors are fabricated from a plasticsmaterial. Rotors are typically a flat disc with an annular ring formingan outward portion thereof which is angled upwardly and has aperturestherein for holding a plurality of reaction tubes. The rotor can be adisposable item which is used for a single set of amplifications.

The rotor drive means can be any drive means used for rotor devices inscientific equipment. For example, the drive means can be adirect-coupled AC motor, a DC motor, or an AC motor that drives therotor via a gearbox or pulleys or the like. Preferably, the drive meansis a direct-coupled AC motor, DC motor or stepper motor with the motorexternal to the chamber.

The microwave energy source typically comprises a magnetron that isexternal to the device chamber with energy delivered to reaction vesselsvia a wave guide or any other means for delivering microwave energyknown to those of skill in the art.

The system for determining denaturation of double-stranded DNA can beany system known to those of skill in the art. Typically, the system isa fluorescence detection system which will be described in greaterdetail below. However, the system can also by an infrared measurementsystem comprising a standard commercially-available IR detection elementmounted on the side of the device chamber and focused on the tips of thereaction vessels to monitor vessel temperature. If necessary,compensation can be made for differences between the temperature of areaction mixture at the point of DNA denaturation and the temperature ofthe vessel wall at that point.

The light source and detector for the fluorescence detection systemcomprises standard components known to those of skill in the art. Forexample, the light source can be an LED, a laser light source or ahalogen lamp, with an appropriate filter to provide light of anappropriate wavelength for excitation of the fluorophore in the reactionmixture.

Emitted fluorescence is typically filtered and then measured by aphotomultiplier tube, CCD array, photodiode or CCD camera. The lightsource and detector are advantageously linked to the associated computerwhereby the application of microwave energy is controlled. It will beappreciated by one of skill in the art that on denaturation of doublestranded DNA, there will be a drop in the fluorescence of the reactionmixture at which point application of microwave energy can beterminated. Advantageously, a double-stranded DNA reference standard—forexample, genomic DNA or cDNA—of a known concentration can be included inreaction mixtures for the purposes of monitoring when denaturationoccurs.

Devices according to the invention can optionally include a mechanismfor cooling the device chamber. Such a mechanism typically comprises anair supply to the chamber wherein the air is either at ambienttemperature or less than ambient by passage through or over a coolingmeans.

The fluorescence detector used to monitor denaturation of doublestranded DNA can also be used to monitor the progress of a reaction. Forexample, the level of fluorescence prior to denaturation can be used toassess the amount of DNA synthesised. Devices can further-more includeadditional monitoring equipment such as a spectrophotometer orphotometer. The additional monitoring equipment can be dedicated toassessing the progress of a reaction while the fluorescence detectionsystem acts solely as a denaturation monitor.

As alluded to above, operation of the device at the beginning and end ofthe amplification, and through each denaturation, annealing andpolymerisation cycle is advantageously controlled by an associatedcomputer. The computer can control such operations as:

-   -   rotor start up and speed (any speed greater than 100 rpm is        suitable but typically rotors are rotated ate 500 rpm);    -   the annealing and polymerisation temperatures and the time for        each of these steps; the period of application of microwave        energy during the denaturation step;    -   processing of data generated by the denaturation system and any        system used to measure the amount of DNA synthesised;    -   rotor braking; and    -   cooling of the device chamber if necessary such as at the        beginning and end of the amplification.

It will be appreciated that the computer can be used to control anyother equipment or mechanisms associated with the device.

With regard to the second embodiment of the invention, the method isadvantageously carried out using the device according to the firstembodiment. It will be appreciated however that it is not essential thatthe device according to the invention be used for the method which isamenable to adaptation to other devices.

With regard to step (i) of the method, the reaction mixture can be anymixture used for an amplification reaction. Typical reaction mixturesare described, for example in standard reference texts such as PCR: aPractical Approach (M J McPherson et al., Ed's), IRL Press, Oxford,England, 1991, and numerous brochures provided by suppliers ofamplification reagents and consumables.

The term “target nucleic acid strand” is used herein in the context of alinear amplification to denote one of the strands of a double strandedDNA molecule (genomic or cDNA) to which the primer will anneal toprovide a complementary sequence thereto in the amplification reaction.However, the method can be used for non linear amplification of doublestranded DNA in which case the reaction mixture will include a primerfor each strand of the DNA molecule. Exponential amplification of thedouble stranded target will result with repetition of the steps of themethod.

With regard to the reagent for the detection of the denaturation ofdouble stranded DNA which can be included in reaction mixtures, thiswill be a fluorophore required for the optical temperature calibrationor fluorescence detection systems referred to above. The intercalatingfluorophore can be any of those known to persons skilled in the art andinclude ethidium bromide and SYBR™ Green.

The annealing and polymerisation steps can be carried out at any of thetemperatures and for the times used in known amplification procedures.

In current instruments for performing PCR and other linear andnon-linear DNA amplification reactions, energy is supplied to thereaction mixture via the reaction vessel wall (glass or plastic) whichacts as an insulator. This process usually takes 1 to 2 minutes for asingle cycle (55° C.-95° C.-55° C.). In a typical reaction, 30 to 50cycles are required.

The device according to the invention allows a reaction vessel to beheld with its wall at typically 50-65° C. with energy for thedenaturation being supplied to the reaction mixture by energizing themagnetron (typically for several seconds). As the reaction vessel wallis held at the typical annealing temperature of 50-65° C., the mixturereturns to that temperature shortly after application of microwaveenergy is terminated due to the fact that the reaction vessel does notabsorb an appreciable amount of microwave energy. This reduces the cycletime to 6 to 10 seconds.

Because of the fast cycle time possible with the device and method ofthe invention, a PCR amplification of double stranded DNA can beperformed in minutes rather than hours. More importantly, linearreactions for the amplification of a single strand of DNA can beexecuted in 1 to 2 hours. This is not achievable with existing devicesand methods which require 100 to 1,000 cycles taking up to 30 hours. Thedevice and method of the invention thus allow real time assays to bedone at least an order of magnitude faster than currently-availableassays.

Having broadly described the invention, a device will now be exemplifiedwith reference to the accompanying drawing briefly described hereafter.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic cross section of a device according to theinvention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

With reference to FIG. 1, device 1 comprises a chamber 2 having rotor 3which is driven by a stepper motor not shown in the drawing. Chamber 2also includes a radial heater 4 and a fan 5 for distributing heated airthroughout the chamber. Heater 4 and fan 5 are mounted to hinged lid 6of the device, which lid can be pivoted out of the way to gain access torotor 3. Rotor 3 has a plurality of holes for holding reaction vessels,one of which vessels is item 7.

Device 1 also includes a magnetron 8 from which microwaves can bedirected via wave-guide 9 to reaction vessels such as 10 as they passthe microwave emission point 11. A light source 12 is provided forilluminating a reaction vessel as it passes through beam 13. Light 14emitted from reaction vessel 7 passes through filter 15 to be detectedby photomultiplier tube 16.

Device components such as the rotor drive, heater 4, fan 5, magnetron 8,and light source 12, are controlled by a computer not shown in thedrawing.

Operation of the device is as follows. Reaction mixtures are dispensedinto reaction vessels using manual pipettors or automated roboticpippetting means and heated to the annealing temperature via heater 4and fan 5 under the control of the associated computer. Rotor 3 isrotated at greater than 100 rpm under the control of the computer duringthis step and subsequent steps to average reaction vessel temperatures.

On command from the computer to denature double stranded DNA formed inthe reaction mixture, magnetron 8 is activated and microwave energytransferred to reaction mixtures via wave guide 9. At least one reactionmixture contains an intercalating dye such as ethidium bromide or SYBR™Green. The dye is excited by light source 12 and fluorescence measuredby photomultiplier tube 16 after selection of light of the appropriatewavelengths by filter 15.

With denaturation of the double stranded DNA, fluorescence emissiondiminishes and on reaching a present level causes the computer todeactivate magnetron 8.

On termination of application of microwave energy, the reaction mixturequickly returns to the annealing temperature maintained within chamber 2by heater 3 and fan 4. The cooling of the annealing temperature takesonly seconds as the reaction vessel per se is not heated by themicrowave energy—only the reaction mixture is acted on by that energy.

At the annealing temperature, the progress of the reaction can bemonitored by way of a fluorescent probe present in reaction mixtures orby measuring the increased energy of the intercalating referred to abovedye. This monitoring is by way of light source 12, filter 15 andphotomultiplier tube 16. Results can be recorded by the computer.

It will be appreciated by a person of skill in the art that many changescan be made to the device and its use as exemplified above withoutdeparting from the broad ambit and scope of the invention.

The term “comprise” and variants of the term such as “comprises” or“comprising” are used herein to denote the inclusion of a stated integeror stated integers but not to exclude any other integer or any otherintegers, unless in the context or usage an exclusive interpretation ofthe term is required.

The reference to the publications cited in the Background Art section ofthis specification is not an admission that the disclosures constitutecommon general knowledge in Australia.

1. A device for the amplification of DNA in a reaction mixture, thedevice comprising: a heated chamber including a rotor for holding aplurality of reaction vessels for reaction mixtures; a drive means forsaid rotor; a microwave energy source with means for controlled deliveryof said energy to said reaction mixtures; and a system for determiningdenaturation of double-stranded DNA.
 2. The device of claim 1, whereinsaid chamber has a lid or sealable opening for loading the device rotor.3. The device of claim 1, wherein said heating of said chamber is by aninternal heater or by supplying heated air through at least one port ina wall of said chamber.
 4. The device of claim 1, which further includestemperature sensor within the chamber which is linked to an associatedcomputer responsible for controlling the operation of the device.
 5. Thedevice of claim 1, wherein said rotor is fabricated from a plasticsmaterial.
 6. The device of claim 1, wherein said rotor drive means is adirect-coupled AC motor, DC motor or stepper motor.
 7. The device ofclaim 1, wherein said microwave energy source comprises a magnetron thatis external to the device chamber with energy delivered to reactionvessels via a wave guide.
 8. The device of claim 1, wherein said systemfor determining denaturation of double-stranded DNA is a fluorescencedetection system or an infrared measurement system.
 9. The device ofclaim 1, which further includes a mechanism for cooling the devicechamber.
 10. A method for the amplification of a nucleic acid strand,the method comprising the steps of: i) forming a reaction mixturecomprising said target nucleic acid strand, nucleotides, a primer, athermostable nucleic acid polymerase, and, if necessary, a reagent forthe detection of denaturation of double-stranded DNA; ii) incubatingsaid mixture at a temperature which allows synthesis of a nucleic acidstrand complementary to said target nucleic acid strand; iii) denaturingdouble-stranded DNA formed in step (ii) by microwave energisation ofsaid reaction mixture with monitoring of said mixture to determine thedenaturation end point; iv) allowing said reaction mixture to cool to atemperature at which primer anneals to said target nucleic acid strand;and v) repeating steps (ii) to (iv) until a desired level ofamplification is achieved.
 11. The method of claim 10, wherein saidmethod is carried out in a device according to claim
 1. 12. The methodof claim 10, wherein said reagent for the detection of the denaturationof double stranded DNA is a fluorophore.